荷载精子相关抗原9基因短发卡RNA的溶瘤腺病毒ZD55-SPAG9联合放疗对前列腺癌LNCaP细胞的治疗作用

Therapeutic effect of the combined use of oncolytic adenovirus carrying short hairpin RNA targeting sperm-associated antigen 9 with radiotherapy on prostate cancer LNCaP cells

目的观察荷载精子相关抗原9(SPAG9)基因短发卡RNA(shRNA)的溶瘤腺病毒ZD55-SPAG9联合放疗对前列腺癌LNCaP细胞的治疗作用并初步探讨其机制。方法将前列腺癌LNCaP细胞分为4组,分别为磷酸盐对照组(PBS组)、放疗组(10 GY)、病毒组[ZD55-SPAG9,10感染复数(MOI)]、联合组(ZD55-SPAG9+放疗,5 MOI+5 GY);细胞计数试剂盒(CCK-8)法检测各组LNCaP细胞增殖能力的变化;Hoechst-33258染色法检测各组细胞的凋亡;Transwell迁移及侵袭实验检测各组细胞的迁移和侵袭能力;蛋白质印迹法(Western blot)检测各组细胞SPAG9蛋白、凋亡相关蛋白及侵袭相关蛋白的表达。两组间差异的比较采用独立样本t检验,多组间差异比较采用One-way ANOVA分析。结果CCK-8结果显示,联合组LNCaP细胞的存活率为(46.02±2.20)%、病毒组为(56.83±2.40)%(t=12.855,P<0.01)、放疗组为(64.12±2.23)%(t=22.389,P<0.01)、PBS组为(99.63±2.72)%(t=59.407,P<0.01),各治疗组的细胞存活率均低于PBS组,差异有统计学意义(F=1407.384,P<0.01),联合组与单一治疗组比较,LNCaP细胞的存活率更低。Hoechst-33258染色实验结果显示,联合组LNCaP细胞的凋亡率为(55.80±1.49)%、病毒组为(36.42±1.78)%(t=-32.295,P<0.01)、放疗组为(30.01±1.80)%(t=-42.814,P<0.01)、PBS组为(9.30±1.53)%(t=-84.343,P<0.01),各治疗组的细胞凋亡率均高于PBS组,差异有统计学意义(F=2010.396,P<0.01),联合组与单一治疗组比较,LNCaP细胞的凋亡更加明显。Transwell迁移和侵袭实验结果显示,(1)迁移实验:联合组迁移细胞数为(102.60±16.03)个、病毒组为(163.67±11.00)个(t=12.166,P<0.01)、放疗组为(206.47±17.05)个(t=17.192,P<0.01)、PBS组为(304.73±17.16)个(t=33.345,P<0.01),各治疗组迁移细胞数均少于PBS组,差异有统计学意义(F=450.498,P<0.01),联合组迁移细胞数明显少于单一治疗组;(2)侵袭实验:联合组侵袭细胞数为(90.93±13.91)个、病毒组为(145.33±15.31)个(t=10.184,P<0.01)、放疗组为(191.33±13.32)个(t=20.187,P<0.01)、PBS组为(295.47±19.00)个(t=33.645,P<0.01),各治疗组侵袭细胞数均少于PBS组,差异有统计学意义(F=467.586,P<0.01),联合组侵袭细胞数明显少于单一治疗组。Western blot实验结果显示,联合组、病毒组、放疗组、PBS组内,SPAG9蛋白相对表达量分别为0.41±0.07、0.81±0.13、1.41±0.17、1.98±0.10,各治疗组SPAG9的相对表达量均低于PBS组,差异有统计学意义(F=98.785,P<0.01),联合组内SPAG9表达明显低于单一治疗组,差异有统计学意义(t=4.868、9.603,P<0.01);凋亡相关蛋白半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-8在联合组、病毒组、放疗组、PBS组内相对表达量分别为1.51±0.11、1.26±0.05、1.11±0.07、0.64±0.10,各治疗组内Caspase-8的表达高于PBS组,差异有统计学意义(F=56.728,P<0.01),与单一治疗组比较,联合组内Caspase-8的上调更加显著,差异有统计学意义(t=-3.713、-5.499,P<0.05);侵袭相关蛋白E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)在联合组、病毒组、放疗组、PBS组内相对表达量分别为(1.44±0.05、0.26±0.06)、(1.23±0.05、0.56±0.01)、(0.91±0.06、0.79±0.07)、(0.25±0.04、1.21±0.07),各治疗组内E-cadherin表达高于PBS组,Vimentin表达低于PBS组,差异有统计学意义(F=287.276、138.020,P<0.01),与单一治疗组比较,联合组内E-cadherin的上调和Vimentin的下调更加显著,差异有统计学意义(t=-5.017、8.386,P<0.01;t=-10.982、10.034,P<0.01)。结论ZD55-SPAG9联合放疗可以显著抑制LNCaP细胞的增殖、迁移和侵袭能力,并促进其凋亡,效果优于单一治疗;机制可能是上调Caspase-8诱导肿瘤细胞凋亡和抑制肿瘤细胞的上皮-间充质转化进程有关。

Objective To observe the therapeutic effect of the combined use of oncolytic adenovirus carrying short hairpin RNA targeting sperm-associated antigen 9(SPAG9)with radiotherapy on prostate cancer LNCaP cells and to initially explore its mechanism.Methods LNCaP prostate cancer cells were divided into 4 groups for intervention,namely:phosphate belanced solution control group(PBS group);radiotherapy group(10 GY);Oncolytic adenovirus group[ZD55-SPAG9,10 multiple infections(MOI)];Combination group(ZD55-SPAG9+radiotherapy,5 MOI+5 GY);cell counting kit-8(CCK-8)was used to detect the changes of LNCaP cell proliferation in each group;Apoptosis was detected by Hoechst-33258 staining;Transwell Migration and Invasion Test was used to detect the ability of cell migration and invasion in each group;The expression of SPAG9 protein,apoptosis-related protein and invasion-related protein were detected by Western blotting.The difference between the two groups was compared by independent sample t-test,and the difference between multiple groups was analyzed by One-way ANOVA.Results CCK-8 results showed that the survival rate of LNCaP cells in the combination group was(46.02±2.20)%,the oncolytic adenovirus group was(56.83±2.40)%(t=12.855,P<0.01),the radiotherapy group was(64.12±2.23)%(t=22.389,P<0.01),and the PBS group was(99.63±2.72)%(t=59.407,P<0.01).The survival rate of cells in each treatment group was lower than that in the PBS group,the difference was statistically significant F=1407.384,P<0.01),and the survival rate of LNCaP cells was lower in the combined group than in the single treatment group.The results of the Hoechst-33258 staining experiment showed that the apoptosis rate of LNCaP cells in the combination group was(55.80±1.49)%,the oncolytic adenovirus group was(36.42±1.78)%(t=-32.295,P<0.01),the radiotherapy group was(30.01±1.80)%(t=-42.814,P<0.01),and the PBS group(9.30±1.53)%(t=-84.343,P<0.01)%.The cell apoptosis rate in each treatment group was higher than PBS,with differences Statistical significance(F=2010.396,P<0.01).compared with the single treatment group,the apoptosis of LNCaP cells in the combination group was more obvious.Transwell migration and invasion experiment results:(1)migration experiment:the number of migrating cells was(102.60±16.03)in the combination group,(163.67±11.00)in the oncolytic adenovirus group(t=12.166,P<0.01),and(206.47±17.05)in the radiotherapy group(t=17.192,P<0.01),and(304.73±17.16)in the PBS group(t=33.345,P<0.01).The number of migrated cells in each treatment group was less than that in the PBS group,the difference was statistically significant(F=450.498,P<0.01).The number of migrating cells in the combination group was significantly reduced compared with that in the single treatment group.(2)invasion experiment:the number of invasive cells in the combination group was(90.93±13.91),that in the oncolytic adenovirus group was(145.33±15.31,t=10.184,P<0.01),that in the radiotherapy group was(191.33±13.32,t=20.187,P<0.01),and that in the PBS group was(295.47±19.00,P<0.01),the number of invasive cells in each treatment group was less than that in the PBS group,and the difference was statistically significant(F=467.586,P<0.01),and the number of invasive cells in the combination group was significantly lower than that in the single treatment group.Western blotting experimental results showed that the grayscale value of the internal parameter protein was 1.Within the combination group,oncolytic adenovirus group,radiotherapy group,and PBS group,the relative expression of SPAG9 protein was 0.41±0.07,0.81±0.13,1.41±0.17,1.98±0.10.The relative expression of SPAG9 in each treatment group was lower than that of the PBS group,and the difference was statistically significant(F=98.785,P<0.01),and the expression of SPAG9 in the combination group was higher;The reduction in the single treatment group was more significant,and the difference was statistically significant(t=4.868,9.603,P<0.01);the relative expression levels of the apoptosis-related protein cysteinyl aspartate-specific protease(Caspase)-8 in the combination group,oncolytic adenovirus group,radiotherapy group,and PBS group were 1.51±0.11,1.26±0.05,1.11±0.07,0.64±0.10.The expression of Caspase-8 in each treatment group was clearly increased compared to the PBS group,and the difference was statistically significant(F=56.28),P<0.01).compared with the single treatment group,the increase in Caspase-8 in the combination group was more significant,and the difference was statistically significant(t=-3.713,-5.499,P<0.05);the relative expression levels of invade-related proteins e-cadherin and Vimentin in the combination group,oncolytic adenovirus group,radiotherapy group,and PBS group were(1.44±0.05,0.26±0.06),(1.23±0.05,0.56±0.01),(0.91±0.06,0.79±0.07),(0.25±0.04,1.21±0.07).compared with the PBS group,the expression of e-cadherin was raised and the expression of Vimentin was lowered in each treatment group.The difference was statistically significant(F=287.276,138.020,P<0.01).compared with the single treatment group,the increase in e-cadherin and the decrease in Vimentin within the combination group were more significant,the difference was statistically significant(t=5.017,8.386,P<0.01;t=-10.982,10.034,P<0.01)Conclusion ZD55-SPAG9 combined with radiotherapy can significantly inhibit the proliferation,migration and invasion ability of LNCaP cells and promote their apoptosis.The effect is superior to that of single treatment;the mechanism may be related to increasing Caspase-8 inducing apoptosis of tumor cells and inhibiting the epithelial-mesenchymal transition process of tumor cells.